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Synaptotagmin-4 (SYT4) is a membrane protein that regulates membrane traffic in neurons in a calcium-dependent or calcium-independent manner. In melanocytes, the intracellular free calcium ion (Ca 2+ ) may be important for dendrite growth and melanogenesis. Mammalian melanocytes originating from neural crest cells produce melanins. Therefore, we predicted that SYT4 might play a role in melanogenesis and the dendrite morphology of melanocytes. To investigate whether SYT4 is involved in melanocyte physiology, SYT4 was overexpressed in alpaca melanocytes and B16-F10 cells. The results showed that SYT4 overexpression resulted in a phenotype consistent with melanogenesis and dendrite extension. At the molecular level, SYT4interacted with extracellular regulated MAP kinase (ERK) to decrease p-ERK activity, which negatively regulated CREB expression. Furthermore, cyclic AMP-responsive element-binding protein (CREB) was upregulated and caused the downregulation of the expression of melanogenic regulatory proteins, including microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein-1 (TYRP1), dopachrome tautomerase (DCT), and transient receptor potential melastatin 1 (TRPM1). Intracellular free Ca 2+ promoted the upregulation of calcium/calmodulin dependent protein kinase IV (CAMK4) expression, which phosphorylated CREB (p-CREB). In turn, p-CREB participated in the transcription of MITF. These results demonstrated that SYT4 promoted melanogenesis through dendrite extension and tyrosinase activity, during which the regulation of Ca 2+ influx via the TRPM1 channel was a key factor.Significance of the study: Intracellular Ca 2+ is important for the function and survival of melanocytes and melanoma cells. SYT4 stimulated melanogenesis through calcium.These results provide evidence that SYT4 regulates Ca 2+ influx through TRPM1 to cause melanogenesis and axonal elongation in alpaca melanocytes and further suggesting that the growth and metastasis of melanoma is controlled by the inhibited expression of SYT4 in melanoma cells.

The efficacy of an inactivated foot-and-mouth disease (FMD) vaccine is mainly dependent on the integrity of the foot-and-mouth disease virus (FMDV) particles. At present, the standard method to quantify the active component, the 146S antigen, of FMD vaccines is sucrose density gradient (SDG) analysis. However, this method is highly operator dependent and difficult to automate. In contrast, the enzyme-linked immunosorbent assay (ELISA) is a time-saving technique that provides greater simplicity and sensitivity. To establish a valid method to detect and quantify the 146S antigen of a serotype O FMD vaccine, a double-antibody sandwich (DAS) ELISA was compared with an SDG analysis. The DAS ELISA was highly correlated with the SDG method (R2 = 0.9215, P<0.01). In contrast to the SDG method, the DAS ELISA was rapid, robust, repeatable and highly sensitive, with a minimum quantification limit of 0.06 μg/mL. This method can be used to determine the effective antigen yields in inactivated vaccines and thus represents an alternative for assessing the potency of FMD vaccines in vitro. But it still needs to be prospectively validated by analyzing a new vaccine preparation and determining the proper protective dose followed by an in vivo vaccination-challenge study to confirm the ELISA findings.